INTRACELLULAR CA2-DEPRIVED CELLS( THRESHOLDS THAT DETERMINE SURVIVAL OR DEATH OF ENERGY)

Increase of intracellular ionized or free Ca2+ is thought to play a ce ntral role hi cell death due to ATP depletion, However, concurrently o perative mechanisms of injury that do not require intracellular Ca2+ i ncreases have made it difficult to test this hypothesis or to determin e the concentrations at which intracellular Ca2+ becomes lethal. The p redominant Ca2+- independent mechanism of injury during ATP depletion involves the loss of cellular glycine. This type of damage can be full y inhibited by adding the amino acid exogenously. Using glycine to sup press Ca2+- independent plasma membrane damage, we have examined the e ffect of intracellular Ca2+ elevations on cell viability during ATP de pletion. Madin-Darby canine kidney (MDCK) cells were depleted of ATP b y incubation with a mitochondrial uncoupler in glucose-free medium, Fr ee Ca2+ concentration in the medium was varied between 26 nmol/L and 1 .25 mmol/L in the presence of a Ca2+ ionophore, Measurements with the Ca2+ probes fura-2, furaptra, and fura-2FF showed that intracellular C a2+ was clamped at extracellular levels under these conditions, Cell s urvival during ATP depletion was indicated by viable cells recovered 2 4 hours later, The results show that ATP-depleted cells can sustain hi gh levels of intracellular Ca2+ (100 mu mol/L) for prolonged periods a nd remain viable if plasma membrane damage is prevented by glycine. Ce ll death was observed only when intracellular free Ca2+ was allowed to increase beyond 100 mu mol/L, and this was associated with dramatic n uclear alterations: chromatin condensation, loss of nuclear lamins, an d breakdown of DNA into large 50- to 150-kb fragments. Our studies dem onstrate unexpectedly high resistance of cells to calcium cytotoxicity if glycine that is lost during ATP depletion is restored. In addition , they provide insights into novel mechanisms of nuclear disintegratio n and DNA damage that are triggered when the high thresholds of intrac ellular Ca2+ required for cell death are exceeded.